Intracellular communication via ubiquitin (Ub) signaling impacts all aspects of eukaryotic cell biology and regulates pathways critical to human development and viability. Aberrations or defects in Ub-signaling can result in numerous debilitating diseases including neurodegenerative diseases, infections, and cancer. Despite remarkable progress over the decades, we still have only a rudimentary understanding of the molecular factors and networks of interactions that govern the assembly of the proteins required for regulated Ub transfer and signaling. The ability to intervene in diseases related to Ub-signaling requires a thorough understanding of these pathways at the molecular level. The basic scheme for Ub modification involves the concerted activities and interactions of several different proteins. This proposal focuses on expanding our understanding of a central player in Ub-transfer reactions, the class of enzymes known as Ub-Conjugating Enzymes or E2s. Once thought simply to shuttle Ub to the site of modification, emerging evidence suggests that E2s can play a pivotal role in substrate recognition, determining the nature of Ub modification of a target protein, and even interacting with and influencing the activities of proteins outside the traditional Ub-transfer pathway. There are approximately 40 E2s in the human genome and most are thought to be directly involved in Ub transfer. Very little functional information is available for the majority of these E2s. The Research Plan outlined in this proposal seeks to substantially expand our understanding of E2 function. Using various biochemical approaches, NMR spectroscopy, crystallography, and mass spectrometry, this project seeks to develop a molecular understanding of the factors that govern 1) the intrinsic activity of E2~Ub conjugates toward particular residues, 2) E2~Ub recognition of substrates, and 3) and regulatory interactions of E2~Ub conjugates that regulate proteins outside the main Ub-transfer pathway. We have adapted and developed new tools with which to identify proteins selectively modified by particular E2~Ub conjugates. We also make use of a new in coli expression system that can reconstruct Ub-transfer pathways in E. coli. This system allows us to generate particular E2~Ub conjugates and investigate their activity in cells without complications from competing Ub-transfer reactions. The new knowledge we seek to obtain will have a significant impact our understanding of human E2 structure and function and greatly accelerate biological research involving ubiquitylation and ubiquitin signaling. PUBLIC HEALTH RELEVANCE: Ubiquitin (Ub) signaling in cells is critical to pathways involved in human development and viability and defects or aberrations in Ub-signaling can result in numerous debilitating human diseases including neurodegenerative diseases, bacterial and viral infections, and cancer. This study seeks to understand key enzymes involved in Ub transfer, the class of enzymes known as E2 Ub-conjugating enzymes, as remarkably little is known about how most human E2s work, what proteins they might target, or the pathways in which they function. The new knowledge we obtain will greatly facilitate the ability of researchers to identify, test, manipulate, and target specific ubiquitylation events in a cell, hel us understand how bacterial pathogens can hijack Ub pathways, and will generate new ways for researchers to investigate the biological impacts of Ub transfer pathways.